Nucleic acid amplification technology (NAAT) is an invaluable and powerful tool in many areas of research and diagnosis. NAAT techniques allow detection and quantification of a nucleic acid in a sample with high sensitivity and specificity as well as quantitative analysis of nucleic acids in a sample.
Nucleic acid amplification may be used to determine the presence of a particular template nucleic acid in a sample, as indicated by the presence of an amplification product following the implementation of a particular NAAT. Conversely, the absence of any amplification product indicates the absence of template nucleic acid in the sample. Such techniques are of great importance in diagnostic applications, for example, for determining whether a pathogen is present in a sample.
The prior art has described a variety of thermocycling and isothermal techniques for amplification of nucleic acids. Thermocycling techniques, such as the polymerase chain reaction (PCR), use temperature cycling to drive repeated cycles of DNA synthesis leading to large amounts of new DNA being synthesised in proportion to the original amount of template DNA. A number of isothermal techniques have also been developed that do not rely on thermocycling to drive the amplification reaction. Isothermal techniques, which utilise DNA polymerases with strand-displacement activity, have been developed for amplification reactions that do not involve an RNA-synthesis step. Similarly, for amplification reactions that do involve an RNA-synthesis step, isothermal techniques have been developed that may use reverse transcriptase, RNase H and/or a DNA-dependent RNA polymerase (see for example, Nucleic Acid Isothermal Amplification Technologies—A Review. Nucleosides, Nucleotides and Nucleic Acids, Volume 27, Issue 3 Mar. 2008, pages 224-243).
The polynucleic acid produced by the amplification technology employed is generically referred to as amplicon. The nature of amplicon produced varies significantly depending on the NAAT being practised. For example, NAATs such as PCR may produce amplicon which is substantially of identical size and sequence. Other NAATs produce amplicon of very varied size wherein the amplicon is composed of different numbers of repeated sequences such that the amplicon is a collection of concatamers of different length. The repeating sequence from such concatamers will reflect the sequence of the polynucleic acid which is the subject of the assay being performed.
Given that NAATs are of paramount importance in many areas, for example diagnostic applications, there is a continued need in the art to provide NAATs which have improved speed, sensitivity and specificity. The present invention provides simple and cost-effective methods for achieving this goal. Furthermore, the present invention has the advantage that the rate increases achieved by the method of the present invention can counteract amplification rate decreases caused by sequence dependent issues that cause primer designs for a particular NAAT to be sub-optimal. Such rate increases can further lower the cost of an assay based on a particular NAAT as costly alternative means to increase amplification rates can be avoided.